Concentration and purification of viruses from particulate magnetic iron oxide-virus complexes



United States Patent 3,470,067 CONCENTRATION AND PURIFICATION OF VIRUSESFROM PARTICULATE MAGNETIC IRON OXIDE-VIRUS COMPLEXES Joel Warren andAlan L. Neal, Terre Haute, Ind., and David A. Rennels, Pasadena, Calif.,assignors to Chas. Pfizer & 'Co., Iuc., New York, N.Y., a corporation ofDelaware No Drawing. Continuation-impart of application Ser. No.489,076, Sept. 21, 1965. This application Sept. 19, 1967, Ser. No.668,946

Int. Cl. C12k 7/00; A61k 23/00 US. Cl. 195-1.5 4 Claims ABSTRACT OF THEDISCLOSURE The ability of iron oxide to complex with microorganismsleads to useful iron oxide-virus compositions and to an invaluable meansof purifying contaminated water.

CROSS-REFERENCE TO RELATED APPLICATIONS This is a continuation-in-partapplication of Ser. No. 489,076, filed Sept. 21, 1965, now abandoned.

BACKGROUND OF THE INVENTION This invention relates generally to animprovement in the preparation of useful biological vaccines and as anincidental ramification thereto, the purification of contaminated water.

The search for biological vaccines in concentrated form having a highdegree of purity is a never ending one. Moreover, a uniform degree ofpurity will insure a consistent potency which is mandatory in allvaccine preparations. As for the purification of water, one of the mostchallenging problems facing the entire world today is a means of makingwater reusable.

SUMMARY OF THE INVENTION The primary aspect of the process of thisinvention involves the preparation of useful iron oxide-virus vaccinecomplexes. The process comprises contacting a virus suspended in anaqueous medium with a particulate magnetic iron oxide, separating theresulting iron oxide-virus vaccine complex from said medium,dissociating the iron oxide-virus vaccine complex and recovering thevirus thereby released.

The novel process disclosed herein offers three distinct advantages whencompared to prior art methods for producing biological vaccines. Theyare:

(1) an inherent purification step, (2) a simple method forconcentration, and (3) a potency enhancement of the resulting antigens.

It is meant to include by the term virus those viruses which are live aswell as inactivated, i.e. dead or attenuated. The invention isapplicable broadly to viruses of the antigenic type which are capable ofcausing infectious diseases in man and animals. Some of the many virusesof this type which can be subjected to the present process are therabies, mumps, influenza, parainfluenza, vaccinia, equineencephalomyelitis, canine distemper, poliomyelitis,encephalomyocarditis, hog cholera, St. Louis encephalitis, yellow fever,adenovirus, measles, Coxsackie, ECHO, and common cold viruses.

The term aqueous medium contemplates any suitable culture system wellknown to those trained in the art. For example, the following areillustrative of suitable culture systems: avian, simian, and human celltissue or tissue cultures; and nutrient broth cultures.

A second or incidental aspect of the process of this invention relatesto a process for the removal of pathogenic organisms from contaminatedwater which com prises passing said contaminated water throughparticulate iron oxide containing a dispersing material in suflicientamount to allow a suitable percolation rate and recovering the purifiedwater.

Unlike the process concerned with the preparation of biological virusvaccines, this aspect deals with all pathogens, that is, anymicroorganism, bacterium or protozoan that produces disease. Evidently,this aspect is concerned with their removal without regard to kindwhereas the vaccine preparation process is concerned with making certainuseful virus vaccines. It should be understood that similar compositionsfor bacteria and protozoans can be prepared, but their usefulness isquestionable. It is found that the present method of purifying water isfar superior to any currently in use. The existing processes includemicroscreening, filtration, chemical coagulation, powdered carbonadsorption and electrodialysis.

DETAILED DESCRIPTION OF THE INVENTION The process for preparing thevaluable magnetic iron oxide-virus vaccine intermediate of thisinvention may be accomplished in two ways:

(a) cultivating a virus in an aqueous nutrient medium or tissue culturein the presence of particulate magnetic iron oxide or (b) first growingsaid virus in an aqueous medium, inactivating it if desired, and thenadding magnetic iron oxide as a powder or suspension.

If the procedure of method (a) is followed, there appears to be noadverse affect on tissue cultures and said iron oxide seems to beharmless to the cells which continue to multiply in a normal fashion.For instance, con.- centrations of iron oxide between about 0.1 mg. andabout 0.5 mg./ml. in chick embryo fibroblast cultures appear to havelittle or no toxicity for the cells and active growth continues in thepresence of the iron oxide for at least one week. Nine-day oldembryonated hens eggs inoculated with 10 mgs. of Fe O into thechlorioallantoic space develop normally for at least three days.

It is not clear whether the iron oxide-vaccine complex which forms is asimple physical mixture or whether true chemical bonding occurs.However, since actual adsorption is favored by elevated temperatures,there is some indication that a true chemical bond may be formed. In thecase of influenza virus, it is found that adsorption rises rapidly to amaximum efficiency of about 10 mg./ ml. of iron at room temperature withshaking for 30 minutes. It is further found that said adsorption isrelatively little influenced by changes in pH in the range of pH 6-9.

With regard to said particulate magnetic iron oxide, it may be in cubicor acicular form. It is found that the smaller the particle size, themore effective is the adsorption of virus from the suspension. Anytreatment which renders the particles more porous and increases thesurface area, as prior treatment with dilute HCl, provides anenhancement in adsorption efficiency. Best results, however, result froma magnetic iron oxide which has been ground to a fine powder by a ballmilling process. An aqueous suspension of this may be sterilized in anautoclave by a steam pressure of 15-20 lbs/sq. in. for 20 minutes.

For purposes of this invention, when reference is made to the term ironoxide, the compound gamma Fe O is contemplated. Although magnetic ironoxide having the formula Fe O is most preferred, it is possible andwithin the scope of this invention to utilize any form of iron oxidewhich is magnetic in nature, for instance, Fe O FeO or any possiblecombinations thereof, may be efliciently substituted for the preferredgamma Fe O The iron oxide-virus vaccine complexes of this invention canbe efiiciently removed from solution by means of application of amagnetic field or low-speed centrifugation. Continuous flow removal andentrapment of the iron oxide complexes can be accomplished by pullingdown the materials with a magnet.

The resulting iron oxide-virus vaccine complex is quite stable and isnot affected by borate or citrate buffers or organic buffers in general.For instance, the virus cannot be removed from the iron complexes bysodium citrate, formate, borate or glutamate. In addition, the bondrcsists a variety of surface-active agents and solvents.

It has been found, however, that effective dissociation of the ironoxide-vaccine complexes and recovery of the adsorbed viruses can beaccomplished by treatment with a saturated aqueous solution of a sodiumor magnesium salt of an acid selected from the group consisting ofphosphoric, sulfuric and carbonic. Particularly effective dissociatingmedia are saturated solutions of sodium phosphate or sodium bicarbonatewhich result in the release of said viruses which can be subsequentlyisolated after removal of the iron oxide by a magnet or centrifuge.

The novel means of purifying water disclosed herein makes use of thesame basic principle which is inherent in the preparation of theaforesaid biological vaccines. The phenomenon that microorganismscomplex with iron oxide lends itself to water purification as aninventive extension thereof. Of course, the removal of said pathogens issubstantially complete and non-selectivity is an essentialcharacteristic of the process. The fact that the water is purified isimportant and what it removes is secondary. There may be times, however,when a sample of contaminated water must be tested for its level ofcontamination. In such an instance the number of microorganisms whichare removed from an aliquot of test water must be collected and measuredagainst a standard or subjected to a suitable diagnostic determination.

The present disclosure describes a method of permitting a continuousflow filtration of relatively large volumes of fluid and the removal ofthe entrapped microorganisms from the filter bed. The use of glass ormetal cylindrical tubes or columns for filtration purposes through suchmaterials as sand, charcoal, diatomaceous earth etc. is well known andworks equally as well for the iron oxide system disclosed herein. Sincetightly packed iron powder is relatively impervious to the passage ofwater, it is necessary to increase the percolation rate and this isaccomplished by mixing said iron oxide with a dispersing material whichpermits the iron oxide to adsorb and retain microorganisms and othermaterials from suspension while allowing free passage of the fluid atthe same time. It has been found that suitable dispersing materials aresand and diatomaceous earth. Although the amount of dispersing materialadded is not critical, it is generally necessary to add more than 50% byweight of material of the total mixture.

As mentioned earlier, there are instances where the amount and kind ofcontamination in a water sample must be determined. This is carried outby:

(a) applying an aliquot of contaminated water onto a column containing amixture of powdered iron oxide and a dispersing material;

(b) dissociating the iron oxide-pathogen complex which forms;

(c) collecting the adsorbed pathogens in a small volume of eluate; and

(d) measuring the level of contamination.

The iron oxide-pathogen complex can be dissociated chemically with anaqueous solution of the sodium or magnesium salt of phosphoric, sulfuricor carbonic acid followed by collection of the pathogens in the eluateor electrically by applying a low intensity current across the columnand collecting the released pathogens by eluting the column withphysiological saline and collecting the eluate.

The kind and amount of contamination can be determined by suitableprocedures which are well known B Lee in the art. For example,concentration of influenza antigen can be measured by means of ahemagglutination test.

Regarding the electrical dissociation, it is found that application of 1to 10 milliamperes at 40 volts with direct current is most preferred.

The following examples are given by way of illustration and should notbe interpreted as limiting the invention, the scope of which is definedby the appended claims.

EXAMPLE I The following table illustrates the efliciency of magneticiron oxide in removing six different influenza viruses from suspensionin infected allantoic fluid:

Virus Concentration in Original 1 Hemagglutinating units/ml.

All strains are in untreated, freshly harvested egg allantoic fluids.After mixing each virus and Fe O at a concentration of 20 mg./ml.,adsorption is carried out at room temperature on an orbital shaker for30 minutes. At the end of this time the iron oxide-virus complex ispulled to the base of the glass container by a strong magnet and thesupernatant clear fluid removed and tested for its ability tohemagglutinate chick erythrocytes, this being a sensitive indicator ofthe presence of residual virus. It is observed from the above table thatconsiderable virus activity is removed from the supernatant fluid afterremoval of the iron oxide-virus complex from the solution.

EXAMPLE II In a manner similar to the procedure outlined in Example I, apoliomyelitis virus, Sabin Type II, is complexed to iron oxide withcomparable results.

EXAMPLE III DETERMINATION OF OPTIMAL AMOUNT OF F0203 FOR VIRUSABSORPTION Virus Gone. in Adsorption Supernato Virus Gone. in EluatoIronQOxido, mg./ml.:

To infected egg allantoic fluid was added the appropriate amounts ofiron oxide. The mixtures were then shaken for 30 minutes at roomtemperature after which the iron oxide-virus complexes were removed witha magnet and the supernatants discarded. The sediment was then elutedwith 10 percent Na I-IPO -7H O by shaking for 10 minutes, the iron oxideagain removed, and the supernatant fluid titrated for its remainingvirus content.

It is thus clearly shown that adsorption rises rapidly to a maximumefficiency of about 10 mg./ml. and all of the original activity isregained in the eluate from the phosphate treated Fe O -virus complex.

5 EXAMPLE IV The table below demonstrates the effective dissociation ofan influenza PR-8 virus from the corresponding iron oxide complex:

Elution with 10% Na HPO '7H O: Virus concentration Original 1 960Adsorption supernate 30 Physiological saline wash of Fe O +virus 40 5Xconc. extracts in Na HPO 3840 Elution with 9% NaHCO (saturated):

EXAMPLE V The table below summarizes the antibody responses in guineapigs which are vaccinated wtih a single dose of a S-strain polyvalentinactivated influenza vaccine wherein the components were concentratedby adsorption onto iron oxide and then eluted in a concentrate, as inExample IV.

CONCENTRATION OF FIVE INFLUENZA ANTIGENS ON FERRIC OXIDE AND ANTIBODYRESPONSE IN VAC- CINATED GUINEA PIGS I. Virus Titers During VaccineProcessing Virus Strain Material AA P R-8 Jap 170 B-Md. B /La EggHarvest fluid 1 640 2, 560 160 80 320 Monovalent vaccine (orig.) 640 2,560 160 80 320 Supernate from F8203 adsorb 128 128 4 4 64 Supernateconcentrated 5, 120 32,000 640 640 2, 560 Actual concentration based onH 8X 12. 5X 4X 8X 8X II. Vaccine Injected Intramuscularly, 0.5 ml.

Original 2 128 512 32 16 64 Supernate from Fe2O3 25 25 1 1 13 10XConcentrate 1, 024 6, 400 128 128 512 III. Antibody Titers of PooledGuinea Pig Sera 3 Wks. Post-Vaccination with 5 Vaccines Shown Original 510 40 320 10 10 Polyvalent Cone. Supernate 10 20 20 20 10 PolyvalentCone. 10X 40 80 80 10 10 Monovalent Conc. Super- 1 nate 10 10 20 10 10Monovalent Cone. 10X 80 80 80 20 20 IV. Geometric Means of IndividualSerum Titers 15 animals-original 3. 2 8. 2 5.0 2. 5 1. 8 15animals-supernate 2. 1 3. 9 1. 8 3. 1 1. 4 11 animals-10X cone. of

supernate 26. 3 42. 6 15. 2 13. 7 6. 7

V. Protein Concentration, gm/m1.

Ori inal 1,125 760 1, 900 310 360 Monovalent supernatant 375 300 450 105150 10X 1, 325 480 1, 040 300 575 TABLEr-Clltil11l8d VI. Total NitrogenConcentration, grnJml.

Virus Strain Material AA PR-8 .Tap 170 B-Md. B/Lee 609 693 610 620 622594 620 575 58 81 24 44 VII. Iron AnalysisResidual in Vaccine, ugm/ml.

10 Original. 0. 2 0. 2 0. 2 0 2 0. 2 10X 0. 2 0. 2 0. 2 0. 8 2. 0

1 HA Units/0.5 ml. 2 HA Units/dose. 3 HAI Titer-Reciprocal of SerumDilution.

The results shown indicate that it is possible to concentrate and purifyinfluenza virus antigen by this method and produce enhanced levels ofantibody in a. vaccinated host.

RETENTION OF HA TITERS IN INFLUENZA VIRUSES 10X CONOENTRATED BY THEFERRIO OXIDE ADSORPTION PROCEDURE AND STORED AT 5 0. FOR 56 DAYS AAPR-BL .Iap 170 B/Md. B/Lee Original 5, 32, 000 640 640 2, 650 AfterStorage 5, 120 10, 000 640 1, 280 5, 120

EXAMPLE VII The procedure of Example I is repeated wherein the influenzastrains shown are grown in the egg allantoic fluid simultaneously in thepresence of the particulate iron oxide. Substantially the same resultsare obtained indicating no side effects resulted from the presence ofiron oxide during the cultivation stage. Experimentally, a suspension ofiron oxide is inoculated with the virus into the eggs. Controls are alsoprepared containing virus alone. The resulting eggs are incubated for 48hours at 35 C., after which time the virus content of the allantoicfluid in the treated eggs and the control untreated eggs is determined.Comparatively, the yields from both are essentially equivalent.

EXAMPLE VIII A glass column with an inner diameter of 1.6 cm. and 22 cm.in length is fitted at the lower end with a perforated rubber stopper.Directly above the stopper is placed a layer (about 1 cm.) of packedglass wool and on top of this is placed a 4 cm. column composed of amixture of Celite No. 545 1 (3 g.) and magnetic iron oxide (211 mg.). Ontop of the column is placed seas and (0.05 cm.).

A concentration of influenza virus, strain B/ G1 (3200 HA/ 1.0 ml.) isintroduced into the filter column in a 10 ml. volume. After the virusflowed through, it was periodically followed by 10 ml. of aliquots ofphysiological saline solution. Each of these sequential aliquots isseparately collected as numbered effluent fractions and tested for theirconcentration of influenza antigen by means of a hemagglutination test.Results indicated virtually complete retention by the column.

EXAMPLE IX The procedure of Exmaple VIII is repeated wherein sand isused in lieu of Celite with comparable results.

EXAMPLE X The procedure of Example VIII is repeated wherein anequivalent concentration of poliomyelitis virus is used in lieu ofinfluenza virus with comparable results.

EXAMPLE XI The procedure of Example VIH is repeated wherein anequivalent concentration of infectious hepatitis virus is used in lieuof influenza virus with comparable results.

1 Trademark of Johns-Manville Co., for dlatomnceous earth.

7 EXAMPLE Xu The procedure of Example VIII is repeated wherein anequivalent concentration of E. coli bacteria is used in place ofinfluenza virus with comparable results.

EXAMPLE XIII The procedure of Example VIII is repeated wherein anequivalent concentration of Leptospira protozoan 15 used in place ofinfluenza virus with comparable results.

EXAMPLE XIV The procedure of Example VIII is repeated and the influenzavirus is removed from the column in the following manner: an aqueoussolution (20% by wt.) of MgSO; (50 ml.) is added in ml. portions inorder to elute the entrapped antigen. Hemagglutination tests carried outon the eluates indicated virtually complete removal from the column.

EXAMPLE XV The procedure of Example XIV is repeated wherein equivalentamounts of the following 20% by weight aqueous solutions are used inlieu of MgSO MgCO a( 4)2 Na CO Na PO Na SO Equivalent results areobtained.

EXAMPLE XVI What is claimed is:

1. A process for the concentration and purification of viruses whichcomprises contacting a virus-infected aqueous cell, tissue or nutrientbroth virus culture system with particulate magnetic iron oxide untilthe virus adsorbs on or complexes with the said iron oxide; magneticallyremoving the resulting iron oxide-virus complex; eluting the saidcomplex to dissociate it by treatment with a saturated aqueous solutionof a sodium or magnesium salt of phosphoric, sulfuric or carbonic acid;and magnetically removing the dissociated iron oxide.

2. A process as in claim 1 wherein said virus is an influenza virus.

3. A process as in claim 1 wherein said virus is a poliomyelitis virus.

4. A process as in claim 1 wherein said virus is cultivated in anaqueous medium in the presence of said particulate iron oxide.

References Cited UNITED STATES PATENTS 2,125,846 8/1938 Laughlin 210-422,351,160 6/1944 Stone et al. 210-30 XR 2,461,505 2/ 1949 Daniel 167-782,642,514 6/ 1953 Herkenhotf 210-24 2,749,306- 6/ 1956 Coleman 210-242,999,792 9/1961 Segre 167-845 3,078,224 2/ 1963 Schulze et al. 210-303,139,401 6/1964 Hach 210-30 3,197,374 7/1965 Hennessen et al. 167-783,256,152 6/1966- Lampson 167-78 3,351,203 11/1967 Robb 210-222 XRFOREIGN PATENTS 887,339 1/1962 Great Britain. 876,027 8/ 1961 GreatBritain.

S. K. ROSE, Primary Examiner US. Cl. X.R.

